Design,Construction And Application Of Reactive Sulfane Sulfur Detection Probes Based On Red Fluorescent Proteins

Reactive sulfane sulfur(RSS)is the type of compounds that contains at least two sulfur atoms connected by a covalent bond,and one of the sulfur atoms is 0-valent.RSS is diverse in environment and organisms,and can be roughly divided into inorganic RSS and organic RSS.RSS generally has a chain structure and the 0-valent sulfur is both electrophilic and nucleophilic,so it can participate in a variety of reactions including the formation of pyrite,the regulation of Ca2+ metabolism and the persulfidation of protein thiols.In recent years,RSS has been found playing important roles in many life processes,such as inflammation,cardioprotection,neuromodulation,antioxidation,mitochondrial function and energy metabolism,intracellular redox homeostasis,etc.There is also a viewpoint that the direct signal compound in vivo is RSS rather than hydrogen sulfide(H2S).All these indicate that RSS is vital in vivo,so we need to further study the distribution,dynamic change and the functioning mechanism of RSS.However,at present,we lack a reliable method to monitor RSS in vivo.In order to address this challenge,we constructed fluorescent probes based on red fluorescent protein(RFP)mCherry and far-red fluorescent protein(FRFP)mKelly1.Both probes can monitor the level of RSS inside cells in real time.We constructed the probes by mutating two amino acid residues,which are close to the chromophore of RFPs,to cysteine residues.When designing the mutation,the distance between the two cysteines was controlled so that when it reacts with RSS,a polysulfide(-Sn-,n?3)bond can be formed,but not a disulfide bond,which can be formed by reacting with reactive oxygen species.The-Sn-bond is located near the chromophore of RFPs,causing changes of the fluorescence characteristics.The level of RSS can be monitored according to the change of fluorescence spectrum.In this study,we constructed three different fluorescent probes.The first is polysulfides-sensitive red fluorescent protein(psRFP),a derivative mCherry,which can be used to monitor the level of RSS in subcellular organelles in real time.The probe detects the level of RSS by the change of fluorescence ratio of 466 nm/610 nm,effectively avoiding the interference of cellular green fluorescence.We have successfully applied this probe to monitor RSS levels in the cytoplasm of Escherichia coli BL21(DE3)and the cytoplasm and mitochondria of Saccharomyces cerevisiae BY4742 in real time.We also applied the probe in FHC and HCT116 cell lines.The second is polysulfides-sensitive far-red fluorescent protein(psFRFP),a derivative of mKelly1.The emission wavelength of psFRFP is located in the far-red zone(630 nm),which is more suitable for in vivo detection than red light.We successfully applied the probe to the cytoplasm of E.coli BL21(DE3)and S.cerevisiae BY4742.The third is that we fused GFP and psFRFP.The GFP-psFRFP can quantify RSS by detecting the fluorescence ratio of 512 nm/630 nm.We determined the standard curve of GFP-psFRFP to HSnH and detected RSS changes in the cytoplasm of S.cerevisiae BY4742.Compared with reported methods,our probes have several obvious advantages.First,they can dynamically monitor cellular levels of RSS in real-time.Second,the psRFP can be used to detect RSS in subcellular organelles.Third,they effectively avoid the interference of intrinsic green fluorescence from live cells.Fourth,GFP-psFRFP can be used for the quantitative detection of RSS in live cells.In summary,we constructed three fluorescent probes to detect RSS levels in live cells.These probes can be used to monitor RSS live cells in real time,locate into subcellular organelles for RSS detection,and quantitatively determine cellular RSS.In this work,we used the probes to study RSS levels in the cytoplasm of E.coli BL21(DE3)and the cytoplasm and mitochondria of S.cerevisiae BY4742.We also applied psRFP in human cell lines.Our probes have added new methods for studying RSS in vivo.